Dihydrothymine lesion in X-irradiated DNA: characterization at the molecular level and detection in cells.

PURPOSE: To study the formation of the dihydrothymine lesion produced in DNA by ionizing radiation in an anaerobic environment. MATERIALS AND METHODS: The dihydrothymine lesion, along with other lesions, was isolated from an X-irradiated aqueous solution of the dinucleoside monophosphate d(TpA) and analysed by correlated two-dimensional nuclear magnetic resonance spectroscopy. The dihydrothymine lesion was obtained by enzymatic digestion of irradiated DNA in the form of modified dinucleoside monophosphates, d(T(d)A), where T(d) stands for dihydrothymidine. Liquid chromatography-tandem mass spectrometry was used to detect the lesion in the DNA of X-irradiated mouse fibroblast cells. RESULTS: The modified dinucleoside monophosphate, d(T(d)pA), fragments by two pathways so that altogether the lesion could be detected using two different sets of tandem mass spectrometry (precursor ion mass/daughter ion mass) values. CONCLUSION: The dihydrothymine lesion is a significant lesion in cells exposed to ionizing radiation in an anaerobic environment.

A theoretical study of the cis-syn pyrimidine dimers in the gas phase and water cluster and a tautomer-bypass mechanism for the origin of UV-induced mutations.

A quantum mechanical study of all cis-syn cyclobutane pyrimidine photodimers including the normal and rare tautomeric forms of bases has been performed using the ab initio method at HF/6-31G(d.p), MP2(fc)//HF/6-31G(d,p) and MP2(fc)/6-31G(d,p) levels. A puckering angle of the cyclobutyl ring and twist angle of pyrimidine rings with respect to each other is well described by these calculations. It is predicted that in the gas phase all photodimers containing the rare imino form of cytosine are more stable than those containing its normal form. The Monte Carlo simulations show that the dimer containing the imino form of cytosine is more stabilized by water cluster than that containing its amino forms. The possible biological significance stems from the fact that the cytosine in the dimer directs the incorporation of adenine in the complementary strand during replicative bypass. Data obtained point to the cytosine tautomerism as a possible mechanism for the origin of UV-induced mutation.

Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.

Assembly of a polytopic membrane protein structure from the solution structures of overlapping peptide fragments of bacteriorhodopsin.

Three-dimensional structures of only a handful of membrane proteins have been solved, in contrast to the thousands of structures of water-soluble proteins. Difficulties in crystallization have inhibited the determination of the three-dimensional structure of membrane proteins by x-ray crystallography and have spotlighted the critical need for alternative approaches to membrane protein structure. A new approach to the three-dimensional structure of membrane proteins has been developed and tested on the integral membrane protein, bacteriorhodopsin, the crystal structure of which had previously been determined. An overlapping series of 13 peptides, spanning the entire sequence of bacteriorhodopsin, was synthesized, and the structures of these peptides were determined by NMR in dimethylsulfoxide solution. These structures were assembled into a three-dimensional construct by superimposing the overlapping sequences at the ends of each peptide. Onto this construct were written all the distance and angle constraints obtained from the individual solution structures along with a limited number of experimental inter-helical distance constraints, and the construct was subjected to simulated annealing. A three-dimensional structure, determined exclusively by the experimental constraints, emerged that was similar to the crystal structure of this protein. This result suggests an alternative approach to the acquisition of structural information for membrane proteins consisting of helical bundles.

Effect of meso-substituents on the osmium tetraoxide reaction and pinacol-pinacolone rearrangement of the corresponding vic-dihydroxyporphyrins.

To investigate the effects of electron-donating and electron-withdrawing substituents upon the reaction of porphyrins with osmium tetraoxide, and the pinacol-pinacolone rearrangement of the resulting diols, a series of meso-substituted porphyrins were prepared by total synthesis. Porphyrins with electron-donating substitutents at the meso-positions gave vic-dihydroxychlorins in which the adjacent pyrrole subunit was predominantly oxidized. No such selectivity was observed in a porphyrin containing a methoxycarbonyl as the electron-withdrawing group, whereas a formyl substituent again resulted in oxidation at the pyrrole unit adjacent to the meso-substituent. Under pinacol-pinacolone conditions, vic-dihydroxy chlorins containing 4-methoxyphenyl or 3,5-dimethoxyphenyl groups at the meso-position showed preferential migration of the ethyl group over the methyl group to give 8-ketochlorins, whereas the diol with an n-heptyl substituent under similar reaction conditions gave both 7- and 8-ketochlorins. In contrast, the diol containing a meso-formyl substituent produced the corresponding 7-ketochlorin exclusively. These results indicate that it is not possible to predict the reactivity of meso-substituted porphyrins in the osmium tetraoxide reaction nor the general substituent migratory aptitudes in the pinacol-pinacolone rearrangement based on simple electronic arguments, most likely because many parameters (e.g., meso-beta-pyrrolic steric crowding and long-range electronic effects) ultimately determine the reactivity. The structural assignments of the porphyrin diols and the keto-analogues were confirmed by extensive (1)H NMR studies; some of the dihydroxychlorins and ketochlorins were found to display unusual features in their (1)H NMR spectra.

The 2-naphthylmethyl (NAP) group in carbohydrate synthesis: first total synthesis of the GlyCAM-1 oligosaccharide structures.

Total syntheses of the GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) oligosaccharide structures: [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-(6-O-SO3Na)-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (1) and [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (2) through a novel sialyl LewisX tetrasaccharide donor are described. Employing sequential glycosylation strategy, the starting trisaccharide was regio- and stereoselectively constructed through coupling of a disaccharide imidate with the monosaccharide acceptor phenyl-6-O-naphthylmethyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside with TMSOTf as a catalyst without affecting the SPh group. The novel sialyl Lewisx tetrasaccharide donor 3 was then obtained by alpha-L-fucosylation of trisaccharide acceptor with the 2,3,4-tri-O-benzyl-1-thio-beta-L-fucoside donor. The structure of the novel sialyl Lewisx tetrasaccharide was established by a combination of 2D DQF-COSY and 2D ROESY experiments. Target oligosaccharides 1 and 2 were eventually constructed through heptasaccharide which was obtained by regioselective assembly of advanced sialyl Lewisx tetrasaccharide donor 3 and a sialylated trisaccharide acceptor in a predictable and controlled manner. Finally, target heptasaccharides 1 and 2 were fully characterized by 2D DQF-COSY, 2D ROESY, HSQC, HMBC experiments and FAB mass spectroscopy.

Chemical synthesis of sulfated oligosaccharides with a beta-D-Gal-(1-->3).

The syntheses of two sulfated pentasaccharides: beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc-(1-->6)]-alpha-D-GalNAc-->OMe (1) and beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc6SO3Na-(1-->6)]-alpha-D-GalNAc-->OMe (2) by using Lewisx trisaccharides 12 and 16 as glycosyl donors are described. Sulfated oligosaccharides 1-2 and intermediate compounds are fully characterized by 2D 1H-1H DQF-COSY and 2D ROESY experiments.

Chemical synthesis of a core 2 branched pentasaccharide containing a carboxylate group.

Design and synthesis of a carboxylate-containing pentasaccahride 1 with the Galbeta(1-4) (Fucalpha1-3)GlcNAcbeta(1-6)[3-[1-carboxymethyl]-Galbeta+ ++(1-3)]GalNAcalpha-OMe sequence, which is obtained through regioselective coupling of the 6-OH of a novel acceptor 9 with Lewis(x) donor 10 catalyzed by NIS-TfOH are described.

A convergent synthesis of trisaccharides with alpha-Neu5Ac-(2 --> 3)-beta-D-gal-(1 --> 4)-beta-D-GlcNAc and alpha-Neu5Ac-(2 --> 3)-beta-D-gal-(1 --> 3)-alpha-D-GalNAc sequences.

The syntheses of three trisaccharides: alpha-Neu5Ac-(2 --> 3)-beta-D-Gal-(1 --> 4)-beta-D-GlcNAc --> OMe, alpha-Neu5Ac-(2 --> 3)-beta-D-Gal6SO3Na-(1 --> 4)-beta-D-GlcNAc --> OMe, and alpha-Neu5Ac-(2 --> 3)-beta-D-Gal-(1 --> 3)-alpha-D-GalNAc --> OBn were accomplished by using either methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-beta-D-glycero-D-g alacto-2-nonulopyranoside)onate or methyl (phenyl N-acetyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-beta-D-gl ycero-D-galacto-2-nonulopyranoside)onate as the sialyl donor. The N,N-diacetylamino sialyl donor appears to be more reactive than its parent acetamido sugar when allowed to react with an disaccharide acceptor under the same glycosylation conditions. The trisaccharides, as well as the intermediate products, were fully characterized by 2D DQF 1H-1H COSY and 2D ROESY spectroscopy.

Total synthesis of sialylated and sulfated oligosaccharide chains from respiratory mucins.

The total syntheses of several complex oligosaccharide moieties that occur in the core structure of sulfated mucins are reported. A trisaccharide acceptor was obtained through regio- and stereoselective sialylation of methyl (6-O-pivaloyl-beta-D-galactopyanosyl)(1-->3)-4,6-O-benzylidene-2-a cetamido-2-deoxy-alpha-D-galactopyranoside with a novel sialyl donor. A tetrasaccharide, pentasaccharide, and hexasaccharide were constructed in predictable and controlled manner with high regio- and stereoselectivity after the successful preparation and employment of a disaccharide donor, trisaccharide donor, disaccharide acceptor, and trisaccharide acceptor building blocks. Finally, a mild oxidative cleaving method was adopted for the selective removal of 2-naphthylmethyl (NAP) in the presence of benzyl groups.

Impact of multicentricity on clinical outcome in patients with T1-2, N0-1, M0 breast cancer.

BACKGROUND: The objective was to determine the impact of multicentric breast cancer on recurrence and survival and to evaluate the current tumor, node, metastasis staging system recommendations for multicentricity in the breast. METHODS: This study included 284 nonpregnant patients with T1-2, N0-1, M0 breast cancer, without previous cancer, who were treated by modified radical mastectomy followed by doxorubicin-based adjuvant chemotherapy. Clinical and pathological data were collected retrospectively and survival was calculated from the date of initial diagnosis using the Kaplan-Meier method. RESULTS: The median follow-up time was 8 years (range, 0.3-24.0), and the median age was 47 years (range, 23-76). The median clinical size of the index tumor was 2.5 cm. In 17% of patients, the clinical nodal status was N1. In 84% of patients, pathology of the index lesion was invasive ductal +/- in situ. Multicentric breast cancer was detected in 60 patients (21%): 30 patients with two lesions, 13 patients with three lesions, and 17 patients with four or more lesions. Locoregional recurrence, contralateral breast cancer, distant metastasis, and survival (disease-specific and disease-free) were similar in both groups of multicentric versus unicentric breast tumors. There was a significant difference between groups in estrogen receptor and axillary lymph node positivity, but these did not contribute significantly to outcome on multivariate analysis. CONCLUSIONS: Multicentricity does not increase the risk of poor outcomes in patients with early-stage breast cancer. This supports the current recommendations of the tumor, node, metastasis staging system that tumor size should be based on the diameter of the largest lesion in patients with multicentric breast cancer.

Three dimensional structure of the seventh transmembrane helical domain of the G-protein receptor, rhodopsin.

PURPOSE: The three dimensional structure of a peptide comprising the sequence of the seventh transmembrane segment of the G-protein coupled receptor, rhodopsin, was determined in solution. METHODS: The sequence of the seventh transmembrane segment of rhodopsin, which contains the NPxxY sequence that is highly conserved among G-protein coupled receptors and lys296 that forms the Schiff base with the retinal, was synthesized by solid phase peptide synthesis. The three dimensional structure was determined in solution by high-resolution nuclear magnetic resonance (NMR). RESULTS: The structure revealed a helix-break-helix motif for this sequence. Two families of structures were observed which differed in the angle between the two helical segments. The sequence of this transmembrane segment overlapped significantly the sequence of a peptide from the carboxyl terminal of rhodopsin, the structure of which was solved previously. The redundant sequence formed a helix in both peptides. It was therefore possible to superimpose the redundant sequence of both peptides and construct a structure for rhodopsin encompassing residues 291-348. CONCLUSIONS: This structure reveals locations of the lys296 and the acylation sites of rhodopsin that are consistent with the known biochemistry of this receptor. This segmentation approach to membrane protein structure provides important structural information in the absence of an X-ray crystal structure of rhodopsin. The approach is expected to be useful for other G-protein coupled receptors.

Structures of the intradiskal loops and amino terminus of the G-protein receptor, rhodopsin.

The intradiskal surface of the transmembrane protein, rhodopsin, consists of the amino terminal domain and three loops connecting six of the seven transmembrane helices. This surface corresponds to the extracellular surface of other G-protein receptors. Peptides that represent each of the extramembraneous domains on this surface (three loops and the amino terminus) were synthesized. These peptides also included residues which, based on a hydrophobic plot, could be expected to be part of the transmembrane helix. The structure of each of these peptides in solution was then determined using two-dimensional 1H nuclear magnetic resonance. All peptide domains showed ordered structures in solution. The structures of each of the peptides from intradiskal loops of rhodopsin exhibited a turn in the central region of the peptide. The ends of the peptides show an unwinding of the transmembrane helices to form this turn. The amino terminal domain peptide exhibited alpha-helical regions with breaks and bends at proline residues. This region forms a compact domain. Together, the structures for the loop and amino terminus domains indicate that the intradiskal surface of rhodopsin is ordered. These data further suggest a structural motif for short loops in transmembrane proteins. The ordered structures of these loops, in the absence of the transmembrane helices, indicate that the primary sequences of these loops are sufficient to code for the turn.

Solution structure of the loops of bacteriorhodopsin closely resembles the crystal structure.

Bacteriorhodopsin is one of very few transmembrane proteins for which high resolution structures have been solved. The structure shows a bundle of seven helices connected by six turns. Some turns in proteins are stabilized by short range interactions and can behave as small domains. These observations suggest that peptides containing the sequence of the turns in a membrane protein such as bacteriorhodopsin may form stable turn structures in solution. To test this hypothesis, we determined the solution structure of three peptides each containing the sequence of one of the turns in bacteriorhodopsin. The solution structures of the peptides closely resemble the structures of the corresponding turns in the high resolution structures of the intact protein.

Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin.

Low resolution electron density maps have revealed the general orientation of the transmembrane helices of rhodopsin. However, high resolution structural information for the transmembrane domain of the G-protein-coupled receptor, rhodopsin, is as yet unavailable. In this study, a high resolution solution structure is reported for a 15 residue portion of the sixth transmembrane helix of rhodopsin (rhovih) as a free peptide. Helix 6 is one of the transmembrane helices of rhodopsin that contains a proline (amino acid residue 267) and the influence of this proline on the structure of this transmembrane domain was unknown. The structure obtained shows an alpha-helix through most of the sequence. The proline apparently induces only a modest distortion in the helix. Previously, the structure of the intradiskal loop connected to helix 6 was solved. The sequence of this loop contained five residues in common (residues 268-272) with the peptide reported here from the rhovih. The five residues in common between these two structures were superimposed to connect these two structures. The superposition showed a root mean square deviation of 0.2 A. Thus, this five residue sequence formed the same structure in both peptides, indicating that the structure of this region is governed primarily by short range interactions.

Inflammatory breast cancer survival: the role of obesity and menopausal status at diagnosis.

No previous studies have evaluated the effect of body size and menopausal status at diagnosis on survival from inflammatory breast cancer (IBC). We evaluated whether obesity and menopausal status had an impact on IBC survival in a cohort of 177 female IBC patients seen from 1974 to 1993 at The University of Texas MD Anderson Cancer Center. Survival time was defined as time from diagnosis until death or censorship at last date of contact. We categorized women by body size by using the National Institutes of Health/National Heart, Lung, and Blood Institute's definitions of obesity as body mass index ((BMI) = weight in kg/(height in m)2) > or = 30, overweight as 25 < or = BMI < 30kg/m2, and normal/lean as BMI < 25 kg/m2. Cox proportional hazards analysis, adjusting for axillary lymph node involvement and chemotherapy protocol, revealed a modifying effect of menopausal status at diagnosis on the association between obesity and IBC survival (P = 0.02). Relative to postmenopausal women, premenopausal women had significantly worse survival (hazard ratio (HR) = 1.51, 95% confidence interval (CI) = 1.03-2.22). After stratifying by menopausal status, premenopausal obese women had non-significantly better survival than their leaner premenopausal counterparts (HR = 0.63, 95% CI = 0.34-1.15) while postmenopausal obese women had significantly worse survival than their leaner counterparts (HR = 1.86, 95% CI = 1.02-3.40). These findings suggest that factors associated with larger body size at diagnosis may contribute to shorter IBC survival among postmenopausal women but not premenopausal women, who were found to have poorer survival regardless of body size.

A novel tumor necrosis factor-alpha inhibitory protein, TIP-B1.

TIP-B1, a novel TNF inhibitory protein, has been identified, purified and characterized from cytosolic extracts of TNF-treated human fibroblasts, and a partial TIP-B1 cDNA clone has been obtained. The (27 kDa pI approximately 4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids), and the nucleotide sequence of the corresponding cDNA clone. TNF-sensitive cells, when exposed to TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Thus, this TIP-B1 treatment effectively makes these cells TNF-resistant. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. TIP-B1 does not interfere with the interactions between TNF and the TNF receptors based on flow cytometric analysis of the cellular binding of biotinylated TNF. These and other data indicate that TIP-B1 is not a soluble TNF receptor, nor an anti-TNF antibody, nor a protease that degrades TNF, yet TIP-B1 functions when added exogenously to cells. Thus, TIP-B1 is not one of the proteins previously reported to be involved in resistance to TNF. The fact that incubation of the newly discovered novel TIP-B1 with TNF-sensitive cells protects them from TNF-induced cell death, including TNF-mediated apoptosis, makes TIP-B1 a candidate for therapeutic modulation of TNF-induced effects.

An efficient synthesis of two monosulfated trisaccharides with the Galbeta1,3GlcNacbeta1,3Galbeta-O-allyl backbone.

The GlcNAcbeta(1-->3) Gal linked disaccharide 7 was synthesized as key building blocks for the construction of target monosulfated trisaccharides 1 and 2 using oxazoline 3 as glycosyl donor promoted by BF3 x Et2O.

An NMR and conformational investigation of the trans-syn cyclobutane photodimers of dUpdT.

Both trans-syn cyclobutane-type photodimers of 2'-deoxyuridylyl (3'-5') thymidine (dUpdT) were formed by deamination of the corresponding trans-syn cyclobutane photodimers of 2'-deoxycytidylyl (3'-5') thymidine (dCpdT) and were examined by 1H-, 13C-, and 31P-nmr spectroscopy. One- and two-dimensional nmr experiments provided a nearly complete assignment of the 1H, 13C, and 31P resonances. Scalar and nuclear Overhauser effect contacts were used to determine the conformation of the deoxyribose rings, exocyclic bonds, cyclobutane rings, and glycosidic linkages. Isomer I (S-type class; CB-; SYN-ANTI) and isomer II (N-type class; CB+; ANTI-SYN) exhibit markedly different conformational features. 31P chemical shifts show that the relative flexibility is dUpdT > isomer II > isomer I. The conformations of these species are very similar to those of other previously examined trans-syn photodimers. Among bipyrimidine photodimers of a given diastereomeric form (i.e., trans-syn I or II), the nmr-derived conformational parameters are nearly invariant, regardless of base substitution pattern. This contrasts with the substituent-dependent variation of cyclobutane ring conformation observed by Kim et al. (Biopolymers, 1993, Vol. 33, pp. 713-721) for an analogous series of cis-syn photodimers. Steric crowding of cyclobutane ring substituents is offered as an explanation for the difference in substituent effects between the families of cis-syn and trans-syn photodimers.

A study of the hydration of deoxydinucleoside monophosphates containing thymine, uracil and its 5-halogen derivatives: Monte Carlo simulation.

An extensive Monte Carlo simulation of hydration of various conformations of the dinucleoside monophosphates (DNP), containing thymine, uracil and its 5-halogen derivatives has been performed. An anti-anti conformation is the most energetically stable one for each of the DNPs. In the majority of cases the energy preference is determined by water-water interaction. For other dimers conformational energy is the most important factor, or both the factors are of nearly equal importance. The introduction of the methyl group into the 5-position of uracil ring most noticeably influences the conformational energy and leads to the decrease of its stabilizing contribution to the total interaction energy. The introduction of halogen atoms increases the relative content of anti-syn and syn-anti conformations of DNPs as compared to the parent ones due to the formation of an energetically more favorable water structure around these conformations. A correlation is observed between the Monte Carlo results for the halogenated DNPs and their experimental photoproduct distribution. The data obtained demonstrates a sequence dependence in the photochemistry of the halogenated dinucleoside monophosphates.